Hi Adrian

          If you have internet access you can see an example uploaded 
          for the second Purdue CD for that on 
          ftp://ftp.cyto.purdue.edu/cdupload/gerhard
          If you have freelance you can look at the whole .pre 
          presentation or otherwise at pages 17 and 18.gif. 

          The major advantage of antibodies over probes is that the 
          bugs remain viable, so you can sort and grow them and you 
          can get simultaneous viability assessment. The disadvantage 
          is that you need to isolate the bug first to make your 
          antibody.

          You will find a reasonable amount of antibodies to clinical 
          relevant bacteria (mostly unlabelled). I have used 
          commercial immunofluorescence kits for counting Clamydia 
          elementary bodies, just adding 10ul to 50ul mashed 
          cellculture diluted 1:100 with PBS after 5 minutes getting 
          good cluster separation, but quite a number of the reagents 
          do not work in flow as they are against extracted antigen 
          like for example parts of flagella only exposed after heat 
          denaturation...

          To set up your equipment for the first time you should use 
          0.5um YG-fluorescent latex with your heatfixed bacteria at 
          1mg/ml propidium iodide and discriminate on green 
          fluorescence. Once you have the beads in the middle of you 
          display you can switch to scatter discrimination. I prefer 
          to look at scatter versus red and scatter versus green (all 
          log) to quickly see clusters coming up.

          Gerhard Nebe-v.Caron
          Unilever Research, Colworth,
          Sharnbrook, Bedfordshire
          GB - MK44 1LQ
          Tel:    +44(0)1234-222066
          FAX:    +44(0)1234-222344
          gerhard.nebe-von-caron at unilever.com





______________________________ Reply Separator _________________________________
Subject: Questions
Date:    08/10/96 00:47
Author:  Adrian_Moris at sratech.com at INTERNET


     Hi folks,

     One of the PI's here at SRA has asked me to start thinking about ways
     to use flow to ID unknown and/or mixed bacterial cultures.  In
     particular, he wants to use surface markers, not DNA probes.  I'm
     fairly new to flow and so far have only been working with mammalian
     cells.  I'd appreciate any advice, especially sources of antibodies,
     advice on looking at things as small as bacteria, etc.  How 'bout it?
     Any bacteria FLOWERS out there who can help me out?

     Oh yeah.  Apologies to those offended at my musical quiz.  I
     personally have enjoyed playing "Sort that tune" as it were, as have
     many of you who replied.  It helps to break up my day and let me think
     more clearly.  But alas, not everyone is like me, so I guess I'll keep
     my notes to the list to more business-related, if mundane, arenas in
     the future.

     Sincerely

     Adrian Moris

     "If you plant ice, you're gonna harvest wind."-JG